Genetic information is encoded on double-stranded deoxyribonucleic acid (DNA) comprising a coding strand and a complementary strand. The genetic information is encoded on the coding strands according to the order the characteristic repeating nucleotide bases are presented. The DNA coding strand comprises long sequences of nucleotide triplets called "codons" which encode specific bits of information. For example, three nucleotides read as ATG (adenine-thyminie-guanine), result in an mRNA signal interpreted as "start translation". Termination codons TAA and TAG are interpreted as "stop translation". Between the start and stop codons, lie the so-called structural gene having codons that define an amino acid sequence.
Synthetic genes offer a number of advantages over their cloned counterparts since they can be designed for optimal expression and flexibility in subsequent manipulations. In addition, they facilitate the study of structure-functional relationships in proteins through the ability to effect mutations through mutagenesis. However, a synthetic gene is a viable option only if it can be synthesized in a reasonably short time in comparison with procedures to isolate the corresponding complementary DNA.
Various methods for making synthetic genes are known. For example, phosphotriester or phosphodiester methods are sometimes used to prepare oligodeoxyribonucleotide fragments. The fragments are then joined together to form longer strands of repeating nucleic acids. U.S. Pat. No. 4,356,270 describes the synthesis and cloning of the somostatin gene comprising about 56 base pairs.
The phosphodiester method of synthesizing genes is disclosed by Brown, E. L. et al, Meth. Enzymol., 68,109 (1979). This method also involves the synthesis of oligonucleotides (oligos) which are subsequently joined together to form the desired nucleic acid sequence.
Methods exist for making genes in large amounts from small amounts. In general, these methods involve the cloning of the gene in an appropriate host system using the techniques of recombinant DNA. In these techniques, the gene is inserted into an appropriate vector which is used to transform a host organism. When the host organism is cultured, the vector is replicated, and hence more copies of the desired gene are produced. Such techniques are disclosed, for example, in Maniatis, T. et al, Molecular Cloning: A Laboratory Manual, Coldspring Harbor Laboratory, pages 390-401 (1982) and aforementioned U.S. Pat. No. 4,356,270.
Current synthetic methods for making genes suffer from several disadvantages. The methods are generally labor-intensive and require a plurality of reaction and isolation steps. It is difficult to make double-stranded DNA sequences greater than 1,000 base pairs in length using such methods because of the numerous side reactions that occur during the chemical synthesis.
The cloning techniques used to produce genes in quantity are also labor-intensive and expensive, requiring multiple steps of editing the gene and the vector into which the gene is inserted, cloning, and separating the cloned gene.
Clearly, the production of double-stranded DNA sequences comprising genetic codes would be enhanced by a method which eliminated the multiple reactions, cloning and isolation steps currently required in the synthetic production of double-stranded DNA.